a) Field of the Invention
The present invention relates to nucleic acid probes that are susceptible to chemical or enzymatic degradation and to assays and methods using such probes in the detection of target nucleic acid sequences in a sample.
b) Description of Related Art
The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the molecular repositories for genetic information. Ultimately, every protein is the result of the information contained in the cell""s nucleic acids. A gene is a segment of DNA that contains the information for a functional biological product such as a protein or RNA. The function of DNA is the storage of biological information, and since cells have typically many thousands of genes, DNA molecules tend to be very large. The total length of all DNA in a single human cell is about two meters, consisting of billions of nucleotides.
In eukaryotic organisms, DNA is largely found in the nucleus of the cell. But protein synthesis occurs on ribosomes in the cytoplasm, hence a molecule other than DNA must carry the genetic message for protein synthesis from the nucleus to the cytoplasm. RNA is found in both the nucleus and cytoplasm, and it carries genetic information from DNA to the ribosome. Several classes of RNAs exist, each having distinct function. Ribosomal RNAs (rRNA) are structural components of ribosomes that carry out the synthesis of proteins. Messenger RNAs (mRNA) are nucleic acids that carry the information from the genes to the ribosome, where the corresponding proteins are made. Transfer RNAs (tRNA) are adapter molecules that translate the information in mRNA into a specific sequence of amino acids. There are also a wide variety of special-function RNAs that carry out additional functions in the cell (Lehninger et al., Principles of Biochemistry, Second Edition, 1993, Worth Publishers, Inc.).
Double-helical DNA consists of two polynucleic acid strands, twisted around each other.
Each nucleotide unit of the polynucleotide strand consists of a nitrogenous base (A, T, C, or G), a sugar deoxyribose, and a phosphate group. The orientation of the two polynucleotide strands is antiparallel in that their 5xe2x80x2 to 3xe2x80x2 directions are opposite. The strands are held together by ydrogen bonds and hydrophobic interactions. The base pairs in DNA consist of purines such as adenine (A) and guanine (G), and pyrimidines such as thymine (T) and cytosine (C). A is paired with T by forming two hydrogen bonds while G is paired with C by forming three hydrogen bonds. This base pair complementarity is an essential feature of the DNA molecule and is due to the size, shape, and chemical composition of the bases. As a result of the geometry of the double helix, a purine must always pair with a pyrimidine. Furthermore, G will always pair with a C, and A will always pair with T. This simple and elegant structure provides for the extraordinary stability of the double helix.
Under the conditions of temperature and ion concentration found in cells, DNA is maintained as a two-stranded structure by the hydrogen bonds of the A-T and G-C base pairs. The duplexes can be melted (denatured into single strands) by heating them (usually in a dilute salt solution of, for example, 0.01 M NaCl) or by raising the pH above 11. If the temperature is lowered and the ion concentration in the solution is raised, or if the pH is lowered, the single strands will anneal, or reassociate, to reconstitute duplexes (if their concentration in solution is great enough). This property is the basis of a technique referred to as nucleic acid hybridization. In a mixture of nucleic acids, only complementary strands will reassociate; the extent of their reassociation is virtually unaffected by the presence of noncomplementary strands. The molecular hybridization can take place between complementary strands of either DNA or RNA or between an RNA strand and a DNA strand.
The use of various hybridization techniques employing oligonucleotide probes to detect genes (and RNA) of interest is well known in the art of molecular biology. Generally, probes are designed so that they hybridize to fragments containing a complementary nucleic acid sequence. The existence and amounts of hybrid formed are detected by measuring radiation (for radioactive probes), enzyme-produced products (for enzyme-labeled probes), fluorescence (for fluorescent-labled probes), and the like, depending on the nature of the signal being used. Various experimental conditions must be calculated to estimate nucleic acid duplex stability of probe-target complexes and to reduce nonspecific (background) binding of probes to non-target DNA or RNA. Due to the many variables that need to be considered when performing hybridization assays, including melting temperature or other denaturation conditions, annealing temperature, salt concentration, pH, and others, the likelihood of nonspecific binding of nucleic acid probes to nontarget nucleic acid sequences is still a major shortcoming when performing various hybridization techniques.
One use of molecular hybridization that has achieved prominence is in situ hybridization. abeled DNA or RNA that is complementary to specific sequences of DNA or RNA in a sample s prepared. Such complementary DNA or RNA is referred to as an oligonucleotide probe. In this assay, oligonucleotide probes are designed to anneal to specific target RNA such as mRNA, or particular native or integrated gene sequences in the DNA. Cells or tissue slices can be briefly exposed to heat or acid, which fixes the cell contents, including the nucleic acid, in place on a glass slide, filter, or other material. The fixed cell or tissue is then exposed to labeled complementary DNA or RNA probes for hybridization. Labeling agents may be radioisotopes such as 32 P, fluorescent dyes, biotinylated nucleotide analogues, antigens or any other commonly performed labeling technique. After a period of incubation, unhybridized labeled DNA or RNA can be removed while the hybridized complexes are detected to reveal the presence and/or location of specific RNA or DNA within individual cells or tissue slices. Although this technique is popular, continued efforts are necessary to improve the sensitivity of the assay and to decrease non-specific binding (background) of labeled probes (Damell et al., Molecular Cell Biology, Second Edition, 1990, Scientific American Books, Inc.).
Another hybridization technique that is commonly used in the art is the hybridization of labeled probes to immobilized nucleic acids. There are many methods available to hybridize labeled probes to nucleic acids that have been immobilized on solid supports such as nitrocellulose filters or nylon membranes. These methods differ in various respects, such as solvent and temperature used; volume of solvent and length of hybridization; degree and method of agitation; use of agents such as Denhardt""s reagent or BLOTTO to block the nonspecific attachment of the probe to the surface of the solid matrix; concentration of the labeled probe and its specific activity; use of compounds, such as dextran sulfate or polyethylene glycol, that increases the rate of reassociation of nucleic acids; and stringency of washing following the hybridization.
In traditional assay methods, several different types of agents can be used to block the nonspecific attachment of the probe to the surface of the solid support. Such agents include Denhardt""s reagent, heparin, nonfat dried milk, and the like. Frequently, these agents are used in combination with denatured, fragmented salmon sperm or yeast DNA and detergents such as SDS. Blocking agents are usually also included in both the prehybridization and hybridization solution when nitrocellulose filters are used. When nylon membranes are used to immobilize the nucleic acids, the blocking agents are often omitted from the hybridization solution, since high concentrations of protein are believed to interfere with the annealing of the probe to its target. In order to minimize background problems, it is best to hybridize for the shortest possible time using the minimum amount of probe, however, it is not always possible to eliminate all nonspecific binding of probes, particularly if the conditions are such that the detectable amount of nucleic acid is low (Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Cold Spring Harbor Laboratory Press).
Nucleic acid hybridization is also the most reliable method for screening cDNA libraries for clones of interest. Large numbers of clones can be analyzed simultaneously and rapidly through the use of nucleic acid probes. Different techniques employ probes of varying length and specification. Homologous probes contain at least part of the exact nucleic acid sequence of the desired cDNA. They can be used in a variety of ways, such as, using a partial clone of an existing cDNA to isolate a full-length clone from a cDNA library. Hybridization with homologous probes is carried out under stringent conditions. Partially homologous probes are used to detect cDNA clones that are related but not identical to the probe sequence. For example, if the same gene has already been cloned from another species, or a related gene has been cloned from the same species, it can be experimentally determined whether the nucleic acid sequence has been sufficiently conserved to allow the screening of a cDNA library by hybridization. It is necessary to establish conditions that will allow the previously cloned gene to be used as a probe for the cDNA of interest, without undue interference from background hybridization resulting in non-specific signal generation. For more detailed information on hybridization assays and conditions, please see Sambrook et al. (supra).
There are several different agents that can be used to block non-specific binding of nucleic acid probes to surfaces in hybridization assays (supra). However, it is desirable to provide improved methods for the use of nucleic acid probes in hybridization techniques that increase the signal-to-noise ratio and decrease non-specific signal generation. The present invention addresses these needs.
The present invention relates to degradable nucleic acid probes and to assays and methods using these probes in the detection of target nucleic acid sequences in a sample. The purpose of the degradation process is to reduce non-specific signal generation.
An assay for detecting a target nucleic acid sequence, employing degradable probes is contemplated by the present invention. Preferably, the assay is a solution-based or an in situ nucleic acid hybridization assay using nucleic acid probes that are susceptible to chemical or enzymatic degradation. The assay comprises (1) hybridizing labeled, degradable nucleic acid probe(s) to a target sequence, creating a target-specific product from the probe(s), (2) degrading or separating the target-complementary region from the labeled region of the probe(s), and (3) detecting the presence of the labeled region.
One aspect of the present invention provides a method for detecting a target nucleic acid sequence in a sample whereby at least one pair of nucleic acid probes anneals to the target nucleic acid. The probes are characterized by two nucleic acid regions which form a terminal probe-probe branch or stem after the base pairing of the probes to the adjacent regions of the target nucleic acid sequence. The regions of the probe which are capable of forming the stem or probe-probe branch as described herein are also referred to as xe2x80x9cstem regionsxe2x80x9d or xe2x80x9cprobe-probe regionsxe2x80x9d. The probes have at least one crosslinking compound positioned within the stem region of at least one of the probes of the probe pair. The stem region of the second probe of the probe pair incorporates a reactant which is capable of forming a covalent bond with the crosslinking compound of the stem region of the first probe. Alternatively, both stem regions may incorporate a crosslinking compound, capable of forming a covalent bond by reaction of two crosslinking compounds, such as by dimerization. The covalent bond occurs after base pairing of the probes to the target nucleic acid sequence, and thereby permanently crosslinks the stem regions of the probe pair to each other. In addition, at least one of the stem regions of the probes contains a detectable moiety or signal-generating moiety bonded to the end of the stem region. The signal is generated and detected after the crosslinking of the stem which in turn occurs after base pairing of the probes to the target nucleic acid sequence. The nucleic acid probes are designed in a manner such that the target-specific hybridization region or target-complementary region of the probe can be separated via a degradation process from the detectable, labeled region. Hence, the remaining portion of the probes consists of the stem and the label. The purpose of this separation is to improve the signal-to-noise ratio by reducing background specific or non-specific signal generation.
Another aspect of the present invention provides nucleic acid probes that comprise a nucleic acid sequence that includes a modified or unmodified nucleic acid region designed to form a probe-probe branch or stem after base pairing of at least two probes to the adjacent regions of a target nucleic acid sequence. The regions of the probe that are capable of forming a stem are also referred to as xe2x80x9cstem regionsxe2x80x9d or xe2x80x9cprobe-probe regionsxe2x80x9d. At least one of the stem regions of the probes contains a detectable moiety, such as a label, bonded to the end of the stem region. In a preferred embodiment of the present invention, the stem region of a probe of a probe pair comprises modified or unmodified purine nucleosides or derivatives, such as modified or unmodified adenine residues and at least one crosslinking compound, while the stem region of the other probe of the probe pair comprises modified or unmodified pyrimidine nucleosides or derivatives that function as reactant for the crosslinking compound. One preferred reactant consists of modified or unmodified thymidine residues. The probes hybridize to adjacent regions of the target sequence and form a probe-probe branch or stem which comprises a three-arm junction. Consequently, after base pairing of the probes to adjacent regions of the target nucleic acid, the stem regions of the probe pair are crosslinked and the terminal stem or probe-probe branch is formed. The covalent crosslink can be induced by photoirradiation or other means. The crosslinked stem is then separated from the target complementary region and subsequently detected. The crosslinking compounds referred to herein are non-nucleosidic, stable, photoactive compounds that comprise coumarinyl derivatives. Examples of crosslinking compounds that react with crosslinking compound reactants such as modified or unmodified pyrimidine nucleosides or derivatives are coumarin derivatives including (1) 3-(7-coumarinyl) glycerol; (2) psoralen and its derivatives, such as 8-methoxypsoralen or 5-methoxypsoralen; (3) cis-benzodipyrone and its derivatives; (4) trans-benzodipyrone; and (5) compounds containing fused coumarin-cinnoline ring systems. All of these molecules contain the necessary crosslinking group located in the right orientation and at the right distance to crosslink with a nucleotide. In addition, all of these molecules are coumarin derivatives, in that all contain the basic coumarin ring system on which the remainder of the molecule is based.
In another embodiment of the present invention, the stem region of a probe pair comprises modified or unmodified purine and/or pyrimidine nucleosides or derivatives and a first crosslinking compound, while the stem region of the other probe of the probe pair comprises modified or unmodified purine and/or pyrimidine nucleosides or derivatives and a second crosslinking compound. After base pairing of the probes to adjacent regions of the target nucleic acid, the stem regions of the probe pair are crosslinked through the reaction (e.g. dimerization) of the crosslinking compounds upon photoirradiation, and the stem or probe-probe branch is formed. The crosslinking compounds referred to herein are non-nucleosidic, stable, photoactive compounds that comprise aryl olefin derivatives. The double bond of the aryl olefin is a photoactive group that covalently crosslinks to a suitable reactant, such as another aryl olefin derivative positioned in the opposite strand of the probe-probe branch. Accordingly, the double bond of the aryl olefin is located in the right orientation and the right distance to crosslink with a non-nucleosidic reactant in the opposite probe-probe region after base pairing of the probes to adjacent regions of the target nucleic acid.
In a particularly preferred embodiment of the invention, the target-specific hybridization region or target-complementary region of the probes is separated via a degradation process from the detectable, labeled region of the probes prior to detection. One objective of this invention is to maintain a covalent connection via the crosslink between the two probe ends that form the labeled probe-probe branch or stem, while removing any portion of the probes up to the site of the crosslink, especially the target-complementary region of the probes. In particular, the target-comlementary region of the probes is to be degraded or separated (e.g. cut away) from the stem of the probes. The methods for degradation or cutting include chemical and enzymatic means, wherein the method of choice depends upon the composition of the probes. The target-complementary region of the probes may comprise ribonucleotides, deoxyribonucleotides, or non-natural substitutes replacing a nucleotide unit(s) in the probes. Thus, the overall design of this system is to provide probes in which the stem region of the probe is resistant to the desired method of degradation or cutting used to separate or remove the target-complementary region of the probe. Following degradation, the separated and detectable portion of the probes consists of the stem and the label which is subsequently measured (vide infra) and quantified. The separation of the target-complementary region from the detectable region of the probes reduces non-specific binding of the probes and thereby improves the signal-to-noise ratio. As a result, background specific or non-specific signal generation is significantly reduced.
In another preferred embodiment of the instant invention, the stem regions of the probes contain a detectable moiety and a capture moiety which are bonded to the end of the stem regions. The detectable moiety may be any signal reporter group, and the capture moiety may be any capture group. In particular, the capture group and signal reporter group may be biotin and fluorescein, respectively. In an alternative embodiment, one or both stem regions of the probes contain detectable moieties which are bonded to the end of the stem regions. The detectable moieties may consist of labels such as fluorophores, radioisotopes, antigens, or enzymes. Furthermore, the labels may be designed such that it is the interaction between the two labels that generates a detectable signal.
In yet another preferred embodiment of the invention, the probes are selected from the roup consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
The detection of target sequences, including but not limited to, full length genes, diagnostic marker genes, expressed sequence tags (ESTs), single nucleotide polymorphisms (SNPs), genomic DNA, cDNA, cccDNA, recombinant genes, and mRNA and rRNA are also encompassed by the present invention.